Post-Reductionist Protein Folding: Determining the In-cell Folding Energy Landsca

后还原论蛋白质折叠：确定细胞内折叠能量景观

• 批准号: 8537946
• 负责人: ANNE GERSHENSON
• 金额: $ 30.38万
• 依托单位: UNIVERSITY OF MASSACHUSETTS AMHERST
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8537946
• 关键词: Address; Aging; Alzheimer' s Disease; Amino Acid Sequence; Amino Acids; Bacillus amyloliquefaciens ribonuclease; Beginning of Life; Biogenesis; Biological; Birth; Blood coagulation; Case Study; Cells; Chemicals; Complex; Crowding; Cystic Fibrosis; Data; Defect; Disease; Disease Attributes; Distal; Employee Strikes; Endoplasmic Reticulum; Environment; Enzymes; Event; Evolution; Explosion; Failure; Family; Fluorescence; Goals; Grant; Immunoglobulins; In Vitro; Inflammation; Knowledge; Label; Lead; Length; Light; Liver Cirrhosis; Maps; Measures; Methods; Molecular Chaperones; Muramidase; Mutation; Nature; Pathway interactions; Pattern; Peptide Hydrolases; Physiological Processes; Plasma; Play; Process; Protein Secretion; Proteins; Psyche structure; Pulmonary Emphysema; Quality Control; Reaction; Research; Ribonucleases; Ribosomes; Role; Scientist; Serine Proteinase Inhibitors; Serpins; Structure; Suicide; Techniques; Testing; Therapeutic; Therapeutic Intervention; Tube; Work; biological research; disease-causing mutation; improved; in vivo; insight; interest; polypeptide; prevent; protein folding; protein misfolding; research study; secretory protein; single molecule

The endoplasmic reticulum (ER) is a folding factory for the cell, able to churn out thousands of secretory proteins per second. Misfolding of a number of these proteins results in diseases ranging from cystic fibrosis to liver cirrhosis. While full-length proteins can be studied in vitro using a myriad of biophysical techniques, thus allowing biophysicists to characterize in detail their folding energy landscapes, studying protein folding in the more physiologically relevant ER environment presents daunting technical obstacles. Using recently developed, powerful single molecule fluorescence techniques on nascent polypeptide chains that contain fluorescently-labeled amino acids, we will determine the conformational evolution as a nascent polypeptide chain elongates in the ER and how conformational space is modulated by interactions with the chaperones and modifying enzymes resident in the ER. The goal is to obtain detailed information on the folding landscape of the growing nascent chain, rivaling the data available in test-tube experiments. Co- and post-translational interactions between the ER-resident proteins and nascent chains likely smooth the folding energy landscape, biasing against misfolded and aggregation-prone intermediates. This remodeling of the folding landscape is particularly important for proteins with high contact order, i.e. many contacts between distal parts of the chain. We will focus on an important family of secreted proteins, the inhibitory serpins, which fold into native structures with high contact order. Intriguingly serpin native states are energetically metastable, poised to change to a different structure. Serpins play crucial biological roles by regulating proteases involved in key physiological processes including blood coagulation and inflammation. A number of serpin mutations, associated with diseases such as liver cirrhosis and emphysema (together called 'serpinopathies'), lead to serpin aggregation in the ER. By studying serpin folding in the ER and comparing it to in vitro folding, we will determine how the folding landscape is altered by interactions with ER-resident proteins and by disease-associated mutations. The results of this research will provide unprecedented insight into how serpins misfold and how their energy landscapes differ in vitro and in vivo. This work will also provide a readily accessible experimental toolbox for scientists studying protein folding in the ER as well as a platform for potential therapeutic strategies to treat serpinopathies and other ER folding diseases.

内质网（ER）是细胞的折叠工厂，每秒能够产生数千种分泌蛋白质。许多这些蛋白质的错误折叠导致从囊性纤维化到肝硬化的疾病。虽然可以使用多种生物物理技术在体外研究全长蛋白质，从而使生物物理学家能够详细描述其折叠能量景观，但在更具生理相关性的ER环境中研究蛋白质折叠存在令人畏惧的技术障碍。使用最近开发的，强大的单分子荧光技术对新生的多肽链，含有荧光标记的氨基酸，我们将确定的构象演变作为一个新生的多肽链在ER中的延长和构象空间是如何调制的相互作用与伴侣和修饰酶驻留在ER。其目标是获得关于不断增长的新生链的折叠景观的详细信息，与试管实验中可用的数据相媲美。ER驻留蛋白和新生链之间的共同和翻译后相互作用可能会平滑折叠能量景观，对错误折叠和易于聚集的中间体产生偏见。这种折叠景观的重塑对于具有高接触顺序的蛋白质特别重要，即链的远端部分之间的许多接触。我们将专注于一个重要的家族分泌蛋白，抑制丝氨酸蛋白酶抑制剂，折叠成天然结构与高接触秩序。有趣的是，丝氨酸蛋白酶原的自然状态在能量上是亚稳态的，随时会改变成不同的结构。丝氨酸蛋白酶抑制剂通过调节参与关键生理过程（包括血液凝固和炎症）的蛋白酶而发挥重要的生物学作用。许多丝氨酸蛋白酶抑制剂突变，与疾病如肝硬化和肺气肿（统称为“丝氨酸蛋白酶抑制剂病”）相关，导致丝氨酸蛋白酶抑制剂在ER中聚集。通过研究丝氨酸蛋白酶抑制剂在内质网中的折叠，并将其与体外折叠进行比较，我们将确定如何通过与内质网驻留蛋白的相互作用和疾病相关突变来改变折叠景观。这项研究的结果将提供前所未有的洞察如何丝氨酸蛋白酶抑制剂错误折叠，以及他们的能量景观如何在体外和体内不同。这项工作还将为研究ER中蛋白质折叠的科学家提供一个易于使用的实验工具箱，并为治疗丝氨酸蛋白酶蛋白病和其他ER折叠疾病的潜在治疗策略提供一个平台。

项目成果

Determining serpin conformational distributions with single molecule fluorescence.

用单分子荧光测定丝氨酸蛋白酶抑制剂构象分布。

• DOI: 10.1016/b978-0-12-385950-1.00016-x
• 发表时间: 2011
• 期刊: Methods in enzymology
• 作者: Mushero,Nicole;Gershenson,Anne
• 通讯作者: Gershenson,Anne

Deciphering protein stability in cells.

破译细胞中蛋白质的稳定性。

• DOI: 10.1016/j.jmb.2013.10.004
• 发表时间: 2014
• 期刊: Journal of molecular biology
• 影响因子: 5.6
• 作者: Gershenson,Anne

All-Atom Simulations Reveal How Single-Point Mutations Promote Serpin Misfolding.

• DOI: 10.1016/j.bpj.2018.03.027
• 发表时间: 2017-07
• 期刊: Biophysical journal
• 影响因子: 3.4
• 作者: Fang Wang;S. Orioli;A. Ianeselli;G. Spagnolli;S. a Beccara;A. Gershenson;P. Faccioli;P. Wintrode